title: Solubilisation of the 5-hydroxytryptamine3 receptor from pooled rat cortical and hippocampal membranes.
abstract: 5-Hydroxytryptamine3 (5-HT3) receptors have been identified in the rat brain using the radioligand [3H]Q ICS 205-930. We report here that these sites have been solubilised from membranes prepared from pooled rat cerebral cortex and hippocampus using various detergents. Of the six detergents tested (1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate, 0.5% deoxycholate, 1% Lubrol, 0.5% digitonin, 1% Triton X-100, and 1% octyl glucoside), deoxycholate (0.5%) yielded the best solubilisation (54.6 +/- 6% of receptor, 70.5 +/- 4% of protein; n = 3). However, most detergents inhibited binding of [3H]Q ICS 205-930 in solution. Binding was found to be optimal after the receptor had been exchanged by gel filtration through Sephadex G-25 into the detergent Lubrol PX (0.05%). Binding of [3H]Q ICS 205-930 to these soluble sites was saturable and specific (Bmax = 46.1 +/- 6 fmol/mg of protein; KD = 0.33 +/- 0.09 nM; n = 4) and was similar to that observed in membranes. Kinetic studies of [3H]Q ICS 205-930 binding demonstrated it to be rapid, with equilibrium being achieved within 15 min at 4 degrees C. The KD determined from the rates of association and dissociation (0.38 nM) agreed well with that determined by saturation analysis. Various antagonists completed for the soluble receptors with a rank order of potency typical for binding at a 5-HT3 receptor site: zacopride (Ki = 0.26 nM) greater than quipazine (0.37 nM) = Q ICS 205-930 (0.33 nM) greater than ICS 205-930 (0.93 nM) greater than GR 38032F (2.2 nM) greater than BRL 24924 (4.1 nM) greater than MDL 72222 (23.4 nM) greater than ketanserin (6,000 nM). The agonists 5-HT and 2-methyl-5-HT also competed for [3H]Q ICS 205-930 binding with high affinity (39.6 and 55.6 nM, respectively). Therefore, we conclude that the 5-HT3 receptor of rat brain has been successfully solubilised, and this should provide a good starting point for purification of the receptor.
authors.affiliation: Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Harlow, Essex, England.
authors.forename: R M
authors.initials: RM
authors.lastname: McKernan
authors.forename: K
authors.initials: K
authors.lastname: Quirk
authors.forename: R G
authors.initials: RG
authors.lastname: Jackson
authors.forename: C I
authors.initials: CI
authors.lastname: Ragan
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002540:Cerebral Cortex / Q000378:metabolism*; D003902:Detergents; D006624:Hippocampus / Q000378:metabolism*; D007700:Kinetics; D008566:Membranes / Q000378:metabolism; D051381:Rats; D011985:Receptors, Serotonin / Q000378:metabolism*; D012995:Solubility
chemical_list: D003902:Detergents; D011985:Receptors, Serotonin